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Molecular Phylogenetics and Evolution 61 (2011) 333–350 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Phylogeography, geographic structure, genetic variation, and potential species boundaries in Philippine slender toads ⇑ Marites Bonachita Sanguila a,1, Cameron D. Siler b, , Arvin C. Diesmos c, Olga Nuñeza a, Rafe M. Brown b a Mindanao State University – Iligan Institute of Technology, Tibanga, Iligan City, Philippines b Biodiversity Institute and Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66045-7561, USA c Herpetology Section, Zoology Division, Philippine National Museum, Rizal Park, Burgos St., Manila, Philippines article info abstract Article history: We investigated phylogeography of Philippine slender toads (genus Ansonia) and used a temporal frame- Received 22 December 2010 work for diversification, statistical tests of alternate topologies, and Bayesian approaches to test previous Revised 22 June 2011 hypotheses concerning dispersal to, and colonization routes within, the southern Philippine island of Accepted 23 June 2011 Mindanao. Two species of Ansonia previously have been documented, with ranges separated by an Available online 3 July 2011 east–west split corresponding to the approximate boundaries of Mindanao’s paleoisland precursors. We present new mtDNA sequence data (1946 bp from genes encoding ND1, 16S rRNA and tRNALeu) for Keywords: 105 Ansonia specimens sampled from 20 localities on Mindanao Island. Our data suggest that Philippine Conservation genetics Ansonia is composed of at least eight, well-supported population lineages, structured into a minimum of Dispersal Evolutionary Significant Units for four highly divergent mtDNA clades. One clade corresponds to Ansonia mcgregori, a range-restricted spe- Conservation (ESUs) cies apparently limited to the distal portion of the Zamboanga Peninsula of western Mindanao. Two mor- Torrent specialist larvae phologically indistinguishable, but genetically divergent, lineages possibly are undescribed cryptic Gene flow species from western Mindanao. We recognize the five remaining lineages as Ansonia muelleri pending Montane endemism data from morphology or bioacoustics that might diagnose separate species among these lineages. Regardless of their species status, the five allopatric lineages of A. muelleri should be viewed as important genetic units for future genetic conservation planning. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction tive or possibly outdated taxonomy (Alcala and Brown, 1998; Bickford et al., 2007; Brown, 2006; Brown and Alcala, 1994; Brown The geologically active islands of the Philippines possess high et al., 2000, 2008; Brown and Stuart, in press; Stuart et al., 2006). levels of endemic vertebrate biodiversity, which is predominantly With robust geographical sampling of genetic material from partitioned hierarchically into (1) Pleistocene Aggregate Island throughout the archipelago, a new group of studies have drasti- Complexes (PAICs; Inger, 1954; Heaney, 1985; Voris, 2000; Brown cally increased species diversity in several clades (e.g., Brown and Diesmos, 2002), (2) individual islands within PAICs, and (3) and Guttman, 2002; Brown et al., 2009; Evans et al., 2003a; montane subcenters of diversity within major landmasses (review: Linkem et al., 2010a, 2010b; Siler et al., 2010; Siler and Brown, Brown and Diesmos, 2009). This nested, highly partitioned nature 2010; Welton et al., 2010a, 2010b). Analyses of rates of species of the archipelago’s fauna has contributed to the recognition of description through time reveal rates of species discovery and the Philippines as a global conservation hotspot, with one of the description unparalleled in the history of Southeast Asian biodi- highest concentrations of land-vertebrate diversity on the planet versity studies (Brown and Diesmos, 2002; Brown et al., 2002, (Mittermeier et al., 1997, 1998, 1999; Reid, 1998; Brooks et al., 2008; Stuart and Bain, 2008; Brown and Stuart, in press; Siler 2002; Brown and Diesmos, 2009). However, the vast majority of et al., 2010, 2011). this diversity is based on species boundaries conceived by tradi- Relative to other parts of the archipelago, the large southern is- tional morphological taxonomy, calling attention to the possibility land of Mindanao has not received the same renewed research fo- of hidden or ‘‘cryptic’’ species diversity masquerading in conserva- cus, due principally to the inaccessibility of its many isolated mountains and logistical obstacles to field work. Much of what is known of the island’s high levels of herpetological diversity and ⇑ Corresponding author. Address: Biodiversity Research Center, 1345 Jayhawk endemism (Taylor, 1920, 1928; Inger, 1954; Brown and Alcala, Blvd., Lawrence, KS 66045, USA. Fax: +1 785 864 5335. 1970; Alcala and Brown, 1998; review: Brown et al., 2000, 2008) E-mail addresses: [email protected] (M.B. Sanguila), [email protected] comes from faunal inventories conducted during the early Euro- du (C.D. Siler). 1 Present address: Fr. Saturnino Urios University, San Francisco St., 8600 Butuan pean exploration (e.g., Boulenger, 1882, 1920; Van Kampen, City, Philippines. 1923; Smith, 1930, 1935), field work conducted in the early 1055-7903/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2011.06.019 334 M.B. Sanguila et al. / Molecular Phylogenetics and Evolution 61 (2011) 333–350 1900s (Taylor, 1915, 1918, 1920, 1922a, 1922b), a single major warrant recognition as species or Evolutionary Significant Units expedition following World War II (Hoogstral, 1951; Inger, 1954), (ESUs) for conservation planning? (2) Are geographic patterns of and field work from the 1960s and 1970s (summarized in Leviton genetic variation consistent with stable, geographically structured, (1963), Brown and Alcala (1970, 1978, 1980)). Subsequent works populations or can we detect the signature of recent geographic or have been scattered, site-specific, and have not yet been synthe- demographic expansion? (3) Do genetic areas of endemism corre- sized in a biogeographic context (Smith, 1993a, 1993b; Amoroso, spond to the isolated montane areas of Mindanao, as would be pre- 2000; Delima et al., 2006, 2007; Nuñeza et al., 2010). Although en- dicted by natural history, larval morphology, and torrent-dwelling demic Mindanao species exemplars have been included in country- larval ecology (Inger, 1960; Brown and Alcala, 1982; Alcala and wide or regional phylogenetic studies (e.g., McGuire and Alcala, Brown, 1998; Inger, 1992)? (4) Can we reject Matsui et al.’s 2000; McGuire and Kiew, 2001; Brown and Guttman, 2002; Evans (2010) recent ‘‘early invasion’’ hypothesis as a general temporal et al., 2003a; Siler et al., 2011), no densely sampled phylogeograph- framework for the invasion of the southern Philippines and subse- ic or population genetic studies of Mindanao endemic vertebrate quent diversification of Philippine Ansonia? (5) Finally, can we re- groups have been conducted. ject hypothesized routes of dispersal along Mindanao’s elongate Here we present a phylogeographic study of the endemic Min- mountain chains as possible corridors for colonization and popula- danao slender toads of the genus Ansonia. Philippine species of tion expansions in order to account for the current distribution of Ansonia historically have been divided into two species, Ansonia Ansonia on the island of Mindanao? muelleri (Boulenger, 1887, from ‘‘Mindanao Island;’’ presumably eastern Mindanao; Inger, 1954, 1960; Alcala and Brown, 1998), 1.1. Geological setting and Mindanao biogeography and Ansonia mcgregori (Taylor, 1922c; from the southern tip of the Zamboanga Peninsula and nearby Basilan Island; Fig. 1). In Because the central portion of the island of Mindanao has been his first review of Philippine Amphibia, Inger (1954) questioned formed by a sequence of collision, accretion, and subduction events the validity of Taylor’s A. mcgregori. However, in later works he that have occurred over the past ten million years (Yumul et al., treated both A. mcgregori and A. muelleri as valid species (Inger, 2003, 2009; Hall, 1996, 1997), it is conceivable that the highly dy- 1960, 1966); other workers have not questioned this perspective namic history of the southern Philippines in part contributed to (Alcala and Brown, 1998; Brown, 2007). Finally, one recent spe- diversification of its fauna. An improved knowledge of the extent cies-level phylogenetic study (Matsui et al., 2010) supported the of land emergence (Lewis, 1997; Hall, 1998), combined with de- distinctiveness of two species of Ansonia on Mindanao and postu- tailed information concerning the timing of landmass collision lated an ancient (20 mya) invasion of the southern Philippines (Yumul et al., 2003; Hall, 1998), suggests possible dispersal routes for the pair of species on Mindanao. for fauna entering the southern portions of the Philippines in its In this paper we ask: (1) do robust genetic sampling and phy- early history. For example, although some components (particu- logeographic analyses support the recognition of only two species larly Zamboanga and extreme eastern Mindanao) may have been of Philippine Ansonia? Alternatively, do phylogeographic patterns land-positive greater than 15 mya, it is clear that they were very indicate the presence of additional divergent lineages that might far apart, and differed radically from today’s configuration (Hall, 1996, 1997, 1998).